The Foxo family of Forkhead transcription factors (Foxos) plays a central role in organismal longevity in invertebrates and is essential for translating environmental stimuli into gene expression programs that confer resistance to oxidative stress in mammals. The main goal of this proposal is to identify the molecular mechanisms by which mammalian Foxos and their protein partners regulate the cellular responses to oxidative stress, as a way to gain insight into the mechanisms that regulate organismal longevity. The Foxo3 member of the Foxo family of transcription factors in mammals is highly expressed in the nervous system. We have shown that in response to oxidative stress stimuli, Foxo3 interacts with the deacetylase Sirt1, a member of the Sir2 family that extends longevity in invertebrates. However, the molecular features that allow Sirt1 and Foxo3 to functionally interact and to control cellular functions in response to oxidative stress are not well understood. To address the question of how the Foxo3/Sirt1 protein complex regulates the responses to oxidative stress in mammalian cells, we propose the following specific aims: 1. To dissect the mechanisms by which Foxo3 and Sirt1 interact in response to oxidative stress 2. To determine the roles of Foxo3 and Sirt1 in response to oxidative stress in neuronal death 3. To determine the mechanisms by which Sirt1 affects Foxo3-dependent gene expression A combination of biochemical and cell biological approaches will be used to develop these aims. Based on recent work in which we identified a series of novel post-translational modifications of Foxo3 that are induced in response to oxidative stress stimuli, we hypothesize that these modifications serve as a 'molecular signature' for the ability of Foxo3 to interact with Sirt1 and control distinct sets of target genes. These experiments will provide critical insight into the mechanisms by which a single transcription factor translates information about oxidative stress into gene expression programs that maintain cellular homeostasis, and will provide a molecular foundation for studying organismal longevity in a mammalian system.